Knowledge of the human genome has given rise to inquiry into individual differences, as well as differences within an individual, as the basis for differences in biological function and dysfunction. Differences as small as single nucleotide polymorphisms (SNPs) or combinations of SNPs can lead to phenotypic differences, and detection of combinations of SNPs can predict the likelihood that an individual will get a specific disease or how an individual will respond to treatment.
For example, most cancers develop from a series of genomic changes, some subtle and some significant, that occur in a small subpopulation of cells. Knowledge of the sequence variations that lead to cancer will lead to an understanding of the etiology of the disease, as well as ways to treat and/or prevent it. An essential first step in understanding genomic complexity is the ability to perform high-resolution sequencing. Therefore, a true understanding of the complexities in either normal or abnormal function will require specific sequence information from large numbers of target nucleic acid molecules.
Bulk sequencing techniques are often not useful for the identification of subtle or rare nucleotide changes due to the many cloning, amplification and electrophoresis steps that complicate the process of gaining useful information regarding individual nucleotides. The ability to sequence and gain information from single molecules obtained from an individual patient is the next milestone for genomic sequencing. However, effective diagnosis and management of important diseases through single molecule sequencing is impeded by lack of cost-effective tools and methods for screening individual molecules.
There have been many proposals to develop new sequencing technologies based on single-molecule measurements, generally either by observing the interaction of particular proteins with DNA or by using ultra high resolution scanned probe microscopy. See, e.g., Rigler, et al., Biotech., 86(3):161 (2001); Goodwin, P. M., et al., Nucleosides & Nucleotides, 16(5-6):543-550 (1997); Howorka, S., et al., Nature Biotech., 19(7):636-639 (2001); Meller, A., et al., Proc. Natl. Acad., 97(3):1079-1084 (2000); Driscoll, R. J., et al., Nature, 346(6281):294-296 (1990). A recent technique employs optical detection in a sequencing-by-synthesis reaction at the single molecule level. Braslavsky, et al., PNAS, 100: 3960-3964 (2003). The present invention provides improvements in sequencing, especially single molecule sequencing.